PROJECT 1: Cellular Senescence and Metabolic Dysfunction ? SUMMARY Kirkland/Tchkonia Aging and obesity, both leading risk factors for type 2 diabetes, are associated with senescent cell accumula- tion in adipose and other tissues. In obese mice, we found senescent cells release insulin-resistance-inducing factors and attract and anchor inflammatory immune cells, amplifying metabolic dysfunction; yet was alleviated by reducing, genetically or with senolytics, senescent cell burden. Our hypothesis is that targeting senescent cells can alleviate age-related metabolic dysfunction and its complications. Whether analogous mechanisms operate in age-related metabolic dysfunction must be tested.. Our goal is to discover and develop interventions that target senescent cells to decrease age-related metabolic dysfunction. Aim 1 is to delineate the molecular mechanisms through which senescent cells cause and are induced by metabolic dysfunction. By testing which metabolically-relevant inducers and mediators of metabolic dysfunction cause cellular senescence we will: as- certain the role of cell type, means through which senescence is induced, and metabolic and inflammatory me- diators on the senescence-associated secretory phenotype (SASP) in adipose tissue cells;.test effects of factors on the SASP in already established adipose tissue senescent cells; elucidate effects of the SASP on adipogenesis and insulin responsiveness; identify causal SASP factors using approaches successful in obesi- ty; and test if fat cells themselves can become senescent-like and acquire a SASP. Aim 2 is to determine cel- lular mechanisms through which senescent cells contribute to metabolic dysfunction. Sequences and depot- dependence of adipose tissue senescent and immune cell accumulation with aging will be examined. We will test the role of different cell types and cells made senescent by various inducers on metabolic dysfunction us- ing a novel senescent cell transplant method to pinpoint mechanisms through which senescent cells accentu- ate metabolic stress. We will knock down key SASP factors in the senescent cells being transplanted and compare effects of transplanted cells in mice vs. rats. Aim 3 is to identify and optimize interventions that allevi- ate senescence-related metabolic dysfunction and its complications. We will test the contributions of adipo- cytes featuring senescence, senescent preadipocytes, endothelial cells, and immune cells to age-related meta- bolic dysfunction using transgenic mice (LOX-ATTAC) from which senescent cells can be removed with spatial and temporal selectivity. We will distinguish roles of p16Ink4a vs. p21Cip1 using INK- and p21-ATTAC mice. The role of cellular senescence in age-related metabolic dysfunction in a second species will be tested in an INK- ATTAC rat model. Effects of new compounds developed by Core B, dosing intervals, and senolytic drug com- binations on age-related metabolic dysfunction will be tested first in a novel adipose tissue explant system and then various aged animals. Metabolic and adipose tissue effects of existing and novel senolytics will be com- pared to effects on bone, the cardiovascular system, and muscle in Projects 2-4.